Macrocyclic polyethers have a widespread use in various areas of science and technology ever since the first preparation of the crown ethers by Pedersen. (Pedersen, C. J. J. Am. Chem. Soc. 1967, 89, 7017.) In the research oriented towards crown ethers a special emphasis has been directed at finding suitable chemical compounds that can selectively recognize and bind cations. Structural modifications of macrocyclic ligands have been performed, where the size of the macrocyclic ring and the type of donor atoms were changed and cation binding properties were investigated (J. J. Christensen, D. J. Eatough, R. M. Izatt, Chem. Rev., 1974, 74, 351-384.; R. D. Hancock, A. E. Martell, Chem. Rev., 1989, 89, 1875-1914.); synthesis of aza-crown ethers (R. A. Schultz, B. D. White, D. M. Dishong, K. A. Arnold, G. W. Gokel, J. Am. Chem. Soc., 1985, 107, 6659-6668.; K. E. Krakowiak, J. S. Bradshaw, D. J. Zamecka-Krakowiak, Chem. Rev., 1989, 89, 929-972.); synthesis of mono and di-lariat aza-crown ethers (G. W. Gokel, D. M. Dishong, R. A. Schultz, V. J. Gatto, Synthesis, 1982, 997-1012.; G. W. Gokel, Chem. Soc. Rev., 1992, 39-47.); synthesis of BiBLE (V. J. Gatto, K. A. Arnold, A. M. Viscariello, S. R. Miller, C. R. Morgan, G. W. Gokel, J. Org. Chem., 1986, 51, 5373-5384.). Various substituents have been introduced and the influence of the structure on the complexing properties of the ligands examined (Lehn, J. M. Supramolecular Chemistry: Concepts and Perspectives, VCH, Weinheim, Germany, 1995.).
Presently, it is possible to define accurately the key factors which influence the affinity of a ligand towards a specific cation, and thus to effectively control its selectivity. (Schneider, H. J.; Yatsimirsky, A. Principles and Methods in Supramolecular Chemistry, Wiley and Sons, Ltd., Chichester, UK, 2000. Steed, J. W.; Atwood, J. L. Supramolecular Chemistry, Wiley & Sons, LTD. Chichester, UK, 2000. Vögtle, F. Supramolecular Chemistry, Wiley, New York, 1991.). For instance, there are several reviews describing interactions between the cations and macrocyclic compounds (J. J. Christensen, D. J. Eatough, R. M. Izatt, Chem. Rev., 1974, 74, 351-384. R. M. Izatt, J. S. Bradshaw, S. A. Nielsen, J. D. Lamb, J. J. Christensen, Chem. Rev., 1985, 85, 271-339.); cation and anion interactions with macrocyclic molecules (R. M. Izatt, K. Pawlak, J. S. Bradshaw, R. L. Bruening, Chem. Rev., 1991, 91, 1721-2085.); interactions of neutral molecules with macrocyclic compounds (R. M. Izatt, J. S. Bradshaw, K. Pawlak, R. L. Bruening, Chem. Rev., 1992, 92, 1261-1354.); or interactions of all above mentioned categories, cations, anions and neutral molecules with macrocyclic crown ethers (R. M. Izatt, K. Pawlak, J. S. Bradshaw, Chem. Rev., 1995, 195, 2529-2586. G. W. Gokel, W. M. Leevy, M. E. Weber, Chem. Rev., 2004, 104, 2723-2750.).
Nevertheless, there are little or no publications describing the use of the particular class of compounds in antitumor therapy. Although the research on potential biological activity of crown ethers is still in its early stages, their potential impact remains large (M. Kralj, Lj. Tu{hacek over (s)}ek-Bo{hacek over (z)}ić, L. Frkanec, Chem Med Chem, 2008, 3:1478-1492 and the references cited therein). From the biological or biomedical point of view, one of the most interesting features of crown ethers is the fact that, due to their ionophoric properties in the membranes, they behave very similarly to natural ionophores, such as gramicidin, valinomycin, nonactin, etc. Naturally occurring ionophores, as metabolites of microorganisms (e.g. Streptomyces sp.), disrupt the flow of ions either into or out of the cells, which dissipate the cellular ion gradients leading to physiological and osmotic stress. Bacteria (particularly gram positive bacteria) are very sensitive to this effect. Since cyclic polyethers clearly discriminate among different ions, they can serve as convenient synthetic model compounds for their biological counterparts and have similar functions (G. W. Gokel, W. M. Leevy, M. E. Weber, Chem. Rev., 2004, 104, 2723-2750). Indeed, crown ethers were found to be toxic in prokaryotic and eukaryotic cellular systems which led to further studies on their potential for being developed as pharmacological agents (W. W. Tso, W.-P. Fung, M. Y. Tso, J. Inorg. Biochem. 1981, 14, 237-244.) It was shown that certain ionophores have antiparasitic activity (e.g. antimalarial or anticoccidial activity) (C. Gumila, M. L. Ancelin, A. M. Delort, G. Jeminet, H. J. Vial, Antimicrob. Agents Chemother. 1997, 41, 523-529.; G. R. Brown, A. J. Foubister, J. Med. Chem. 1983, 26, 590-592.), therefore efforts have been made to prepare efficient crown compounds with potential antiparasitic activity. In addition, certain crown ethers were found to have significant antifungal activity against some wood-decay fungi, phytopathogenic fungi, eumycetes and trichophytons for dermatomycosis. Yagi and co-workers found that among the 26 crown ethers tested 3,5-di-t-butyl-benzo-15-crown-5 showed relatively high activity, while unsubstituted crown compounds or those with a polar substituent were inactive (K. Yagi, V. Garcia, M. E. Rivas, J. Salas, A. Camargo, T. Tabata, J. Incl. Phenom. Macrocycl. Chem. 1984, 2, 179-184.). Tso and co-workers found that substituted 18-crown-6 ethers show different inhibitory effects on the growth of E. Coli (W. W. Tso, W.-P. Fung, M. Y. Tso, J. Inorg. Biochem. 1981, 14, 237-244.; W. W. Tso, W.-P. Fung, Inorg. Chim. Acta 1980, 46, L33-L34). Thereafter, various approaches were developed to prepare crown-based antimicrobial agents. Leevy and co-workers determined minimal inhibitory concentrations of several alkyl-substituted lariat ethers on E. coli, B. subtilis and yeast (W. M. Leevy, M. E. Weber, M. R. Gokel, G. B. Hughes-Strange, D. D. Daranciang, R. Ferdani, G. W. Gokel, Org. Biomol. Chem. 2005, 3, 1647-1652.). The authors proposed the mechanism for toxicity. It depends on the ability of these compounds to transport ions, most probably by inserting and integrating into membrane bilayer and conducting cations as expected for carriers, whereby the side chain length and hydrophobicity play an essential role. Sensitivity to various compounds varied among the tested organisms, depending on the membrane structures. Furthermore, numerous approaches were developed to build more complex synthetic ion transporters with potential biological activity—the channels such as crown ether peptide nanostructures having ion channel or hydrophile activity (G. W. Gokel, W. M. Leevy, M. E. Weber, Chem. Rev., 2004, 104, 2723-2750; E. Biron, F. Otis, J.-C. Meillon, M. Robitaille, J. Lamothe, P. Van Hove, M.-E. Cormier, N. Voyer, Bioorg. Med. Chem. 2004, 12, 1279-1290).
Biological Activity in Mammalian Cells
Immediately after the crown ethers were discovered, their toxic effect to higher organisms was observed. More than 20 years ago various studies have been performed showing toxicity of different cationic ionophores (including crown ethers) in multiple species, such as mice, rats and dogs (K. Takayama, S. Hasegawa, S. Sasagawa, N. Nambu, T. Nagai, Chem. Pharm. Bull. 1977, 25, 3125-3130.; S. C. Gad, W. J. Conroy, J. A. McKelvey, K. A. Turney, Drug Chem. Tox. 1978, 1, 339-353.; R. R. Hendrixon, M. P. Mack, R. A. Palmer, A. Ottolenghi, R. G. Ghirardelli, Toxicol. Appl. Pharmacol. 1985, 8, 451-468.; S. C. Gad, C. Reilly, K. Siino, F. A. Gavigan, G. Witz, Drug Chem. Tox. 1985, 8, 451-468.). These studies have clearly shown that the majority of the ionophores induced neurotoxic effects of reversible pharmacological nature. Crown compounds have also shown neither to be genotoxic nor mutagenic (M. Kralj, Lj. Tu{hacek over (s)}ek-Bo{hacek over (z)}ić, L. Frkanec, ChemMedChem, 2008, 3:1478-1492).
However, although their cytotoxic effects on mammalian cells (including tumor cells) were soon recognized, no systematic studies were performed on the potential antitumor ability of crown ether compounds. The exceptions are functionalized crown ethers synthetically designed to interact with DNA: e.g. to alkylate and/or cleave DNA in order to achieve antitumor activity (M. Kralj, Lj. Tu{hacek over (s)}ek-Bo{hacek over (z)}ić, L. Frkanec, ChemMedChem, 2008, 3:1478-1492). The emphasis was put on the mutual effect of two functionally different parts: e.g. one part carries a DNA intercalating function, while the other has an ability to bind metal ions. Thus, the DNA binding ability of such compounds should be influenced or regulated by metal ions complexation, since metal complexation should lead to the change in the net electric charge along with the change in the ligand conformation (T. Ihara, S. Sueda, A. Inenaga, R. Fukuda, M. Takagi, Supramol. Chem. 1997, 8, 93-111). DNA binding and intercalation studies were performed with different crown compounds having various side arms, such as crown ether linked DNA-intercalators (e.g. acridine or antraquinone derivatives). In these studies the acridine subunit binds to DNA while the crown subunit binds cations which interact with the phosphate DNA backbone, thus stabilizing the complex (R. Fukuda, S. Takenaka, M. Takagi, J. Chem. Soc., Chem. Commun. 1990, 1028-1030). Furthermore, crown ether derivatives of actinomycin D (AMD) containing benzo-15-crown-5 and benzo-18-crown-6 groups attached via amide bonds were also described and tested in human and mouse tumor model systems (L. Karawajew, E. N. Glibin, V. Ya. Maleev, G. Czerwony, B. Dörken, D. B. Davies, A. N. Veselkov, Anti-Cancer Drug Des. 2000, 15, 331-338.; N. P. Yavorskaya, I. S. Golubeva, I. Yu. Kubasova, A. V. Ovchinnikov, N. G. Plekhanova, E. N. Glibin, Pharm. Chem. J. 2001, 35, 305-307.).
Some functionalized crown compounds were designed to covalently modify (alkylate) and cleave DNA in the ion-regulated manner. Two groups of researchers developed compounds that alkylate and cleave DNA and also stops the tumor cell growth (K. Brandt, R. Kruszynksi, T. J. Bartczak, I. Porwolik-Czomperlik, Inorg. Chim. Acta 2001, 322, 138-144.; M. M. McPhee, J. T. Kern, B. C. Hoster, S. M. Kerwin, Bioorg. Chem. 2000, 8, 98-118.; M. M. McPhee, S. M. Kerwin, Bioorg. Med. Chem. 2001, 9, 2809-2818). For example, compounds having aziridinyl groups introduced in the crown-bearing cyclotriphosphazene, such as tetraaziridinyl-lariat ethers, were tested for in vitro antitumor activity in an investigational AIDS-related lymphoma screening. As a result of synergistic effects from the interaction of metal and (di)alkylating aziridinyl with DNA, this compound showed remarkable cytostatic activity. The DNA damage stops cell proliferation, making this compound a cytostatic drug (K. Brandt, R. Kruszynksi, T. J. Bartczak, I. Porwolik-Czomperlik, Inorg. Chim. Acta 2001, 322, 138-144).
Propargylic sulphone-armed lariat crown ethers and bis(propargylic) sulfone crown ethers were prepared in order to causal connect molecular recognition of specific alkali metal ions to DNA damage under conditions of elevated alkali metal ion levels reported to exist in tumor cells. These compounds were tested against tumor cell growth inhibition at the National Institute of Health, National Cancer Institute, some of them showing significantly more pronounced DNA cleavage and cytotoxic activity compared to the non-crown ether analogues (M. M. McPhee, J. T. Kern, B. C. Hoster, S. M. Kerwin, Bioorg. Chem. 2000, 8, 98-118.; M. M. McPhee, S. M. Kerwin, Bioorg. Med. Chem. 2001, 9, 2809-2818). Also, platinum-based DNA-binding/alkylating agents containing crown ether moiety were prepared and tested for potential antitumor activity. The antitumor effect of platinum compounds is ascribed to the reaction of platinum atom with nucleophilic DNA sites, whereby major adducts and intrastrand cross-links are formed by binding of cisplatin to two neighbouring guanines. An example of potential platinum antitumor compound having platinum atoms linked through a spacer or pendant coordinating groups is 18-crown-6-tetracarboxybis-diammineplatinum(II). Its antitumor activity is tested in different tumor models and in general corresponds to cisplatin activity in cisplatin-sensitive as well as in cisplatin-resistant cells. Moreover, its toxicity in vivo is considerably lower (S. Frühaufand, V. J. Zeller, Cancer Res. 1991, 51, 2943-2948.).
Above mentioned examples show attempts to prepare potential antitumor and other biologically active compounds in which crown ethers, as a part of the molecule, facilitate or enhance the inherent feature (mode of action) of the other part(s) of the same compound. However, as previously mentioned, no systematic study was performed on the antitumor potential of non-functionalized crown compounds. Interestingly, in none of the experiments described above crown ethers were tested in parallel with the crown ether substituted derivatives in order to assess their independent activity. In general, there are limited reports about antiproliferative activity of crown ethers in mammalian cells. Possible antiproliferative/antitumor activity of conventional crown ethers and their derivatives was recently studied and compared to valinomycin. Various derivatives of 18-crown-6 compounds were chosen, as most frequently studied crown ether compounds, along with one 15-membered ring derivative and two derivatives with larger macrocyclic rings: dibenzo-24-crown-8 and dibenzo-30-crown-10. All of them, except the crown ether with the smallest ring, preferred potassium over sodium ion complexation. The results clearly revealed that crown ethers have remarkable tumor-cell growth inhibitory activity and that this activity strongly correlates to both the type of hydrophilic cavity (the size and the nature of donor atoms) and the characteristics of surrounding hydrophobic ring (M. Marjanovi{hacek over (c)}, M. Kralj, F. Supek, L. Frkanec, I. Piantanida, T. {hacek over (S)}muc, Lj. Tu{hacek over (s)}ek-Bo{hacek over (z)}ić, J. Med. Chem. 2007, 50, 1007-1018.). This work clearly demonstrated the great importance of substituents to this effect, which is enhanced by increasing the hydrophobicity possibly due to requirements for membrane insertion. Nevertheless, neither the lipophilicity nor the K+ binding constants exhibit a linear relationship to antiproliferative activity, indicating that a combination of various molecular properties determine their biological activity.
Although numerous examples of adamantane derivatives of crown ethers are known (A. P. Marchand, K. A. Kumar, A. S. McKim, K. Mlinari{hacek over (c)}-Majerski, G. Kragol, Tetrahedron, 1997, 53, 3467-3474.; K. Mlinari{hacek over (c)}-Majerski, G. Kragol, Tetrahedron, 2001, 57, 449-457.; K. Mlinari{hacek over (c)}-Majerski, A. Vi{hacek over (s)}njevac, G. Kragol, B. Koji{hacek over (c)}-Prodi{hacek over (c)}, J. Mol. Struct., 2000, 554, 279-287.; A. P. Marchand, S. Alihod{hacek over (z)}ić, A. S. McKim, K. A. Kumar, K. Mlinari{hacek over (c)}-Majerski, T. {hacek over (S)}umanovac, S. G. Bott, Tetrahedron Lett., 1998, 39, 1861-1864.; A. P. Marchand, K. A. Kumar, A. S. McKim, S. Alihod{hacek over (z)}ić, H.-S. Chong, K. Krishnudu, M. Takhi, K. Mlinari{hacek over (c)}-Majerski, G. Kragol, T. {hacek over (S)}umanovac, Kem. Ind., 2001, 50, 129-138.; O. A. Raevski, V. V. Tka{hacek over (c)}ev, L. O. Amovman, I. O. Umarova, A. F. Solomonov, T. N. Kundra, A. A. {hacek over (C)}ajkovskaja, Izv. Akad. Nauk SSSR Ser. Khim., 1984, 2028-2036.; A. A. {hacek over (C)}ajkovskaja, T. N. Kundra, A. M. Pin{hacek over (c)}uk, Zh. Ob. Khim., 1987, 57, 671-675. A. A. {hacek over (C)}ajkovskaja, T. N. Kundra, A. M. Pin{hacek over (c)}uk, Zh. Org. Khim., 1989, 25, 2000-2003.; K. Naemura, T. Mizo-oku, K. Kamada, K. Hirose, Y. To be, M. Sawada, M. Takai, Tetrahedron:Asymmetry, 1994, 5, 1549-1558.; K. Hirose, J. Fuji, J. Kamada, Y. To be, K. Naemura, J. Chem. Soc., Perkin Trans. 2, 1997, 1649-1657.; Y. To be, Y. Tsuchiya, H. Iketani, K. Naemura, K. Kobiro, M. Kaji, S. Tsuzuki, K. Suzuki, J. Chem. Soc., Perkin Trans. 1, 1998, 485-494.: S. Eguchi, H. Miyake, A. Gupta, T. Okano, Heterocycl. Commun., 1998, 4, 217-226) there are only few reports on adamantane aza-crown ethers (D. Ranganathan, V. Haridas, I. L. Karle, Tetrahedron, 1999, 55, 6643-6656.; K. Mlinari{hacek over (c)}-Majerski, T. {hacek over (S)}umanovac Ramljak, Tetrahedron, 2002, 58, 4893-4898). The syntheses of the compounds of type II, which are the subject matter of the present invention, and their extraction and binding properties towards alkali metal cations were described in our earlier report (K. Mlinari{hacek over (c)}-Majerski, T. {hacek over (S)}umanovac Ramljak, Tetrahedron, 2002, 58, 4893-4898.), but no biological activity was investigated up to now.